We have continued to explore the role that reactive oxygen species (ROS)play in signal transduction pathways. We have shown that a variety of growth factors and cytokines induce the generation of ROS following ligand binding. Our studies suggest that the pathway leading to ROS generation involves the activation of the small GTP-binding proteins ras and rac1. This has led us to explore fully the role of these proteins, and in particular, their role in vascular biology. We have also explored the role that ROS play in apoptosis, and more recently in replicative senescence. Our current focus is on how small the cellular redox state regulates biological events. In the last year we have been able to demonstrate that mitochondrial oxidants stimulate a signaling pathway that regulates glycogen synthase kinase 3. We have proposed that mitochondrial oxidants may function physiologically to regulate cellular metabolism. In this scenario the release of mitochondrial oxidants stimulated by an increase in metabolism would activate the above pathway , that in turn , would feedback and inhibit metabolism, and hence oxidant generation. In addition, we have attempted to explore the relationship between senescence and cardiovascular disease. We were able to show that the small GTPase ras can induce senescence in primary human fibroblasts through an increase in mitochondrial oxidants. These results help stimulate a growing interest in the regulation of mitochondrial oxidant production. We were also able to show that homocysteine, a known cardiac risk factor, can accelerate the rate of endothelial senescence. Interestingly, preliminary evidence suggests that senescent cells colocalize with atherosclerotic plaque in human arterial segments in vivo. Finally, we have attempted to identify direct targets of oxidant stress. We have developed a new assay to allow for the rapid isolation and subsequent identification by mass spectroscopy of proteins in which glutathione has been added in a covalent fashion. We are attempting to understand the physiological relevance of glutathiolation in various signaling pathways. - reactive oxygen species (ROS), GTP-binding proteins, apoptosis